INSTRUMENTOS DE EVALUACIÓN Y RECOLECCIÓN DE LA DATA

Overview

Ect2 is a member of the human Dbl family of guanine nucleotide exchange factors (RhoGEFs) that serve as activators of Rho family small GTPases. Although Ect2 is one of at least 25 RhoGEFs that can activate the RhoA small GTPase, cell culture studies using established cell lines determined that Ect2 is essential for mammalian cell cytokinesis and proliferation. To address the function of Ect2 in normal mammalian development, we performed gene targeting to generate Ect2 knockout mice. The heterozygous Ect2+/- mice showed normal development and lifespan, indicating that Ect2 haplodeficiency was not deleterious for development or growth. In contrast, Ect2-/- embryos were not found at birth or post-implantation stages. Ect2-/- blastocysts were recovered at embryonic day 3.5 but did not give rise to viable outgrowths in culture, indicating that Ect2 is required for peri-implantation development. To further assess the importance of Ect2 in normal cell physiology, we isolated primary fibroblasts from Ect2fl/fl embryos (MEFs) and ablated Ect2 using adenoviral delivery of Cre recombinase. We observed a significant increase in multinucleated cells and accumulation of cells in G2/M phase, consistent with a role for Ect2 in

1Chapter adapted from published paper: “The ect2 rho Guanine nucleotide exchange factor is essential for early mouse development and normal cell cytokinesis and migration” Cook, D. R., Solski, P. A., Bultman, S. J., Kauselmann, G., Schoor, M., Kuehn, R., Friedman, L. S., Cowley, D. O., Van Dyke, T., Yeh, J. J., Johnson, L., Der, C. J. Genes and

cytokinesis. Ect2 deficiency also caused enlargement of the cytoplasm and impaired cell migration. Finally, although Ect2-dependent activation of RhoA has been implicated in cytokinesis, Ect2 can also activate Rac1 and Cdc42 to cause growth transformation. Surprisingly, ectopic expression of constitutively activated RhoA, Rac1, or Cdc42, known substrates of Ect2, failed to phenocopy Ect2 and did not rescue the defect in cytokinesis cause by loss of Ect2. In summary, our results establish the unique role of Ect2 in development and normal cell proliferation.

Introduction

Rho family small GTPases are regulators of diverse cellular processes that include cell proliferation and survival, actin organization and cell shape, polarity and movement, and endocytosis and exocytosis [6, 49]. There are 20 human Rho GTPases, with RhoA, Rac1 and Cdc42 the best-characterized members. Rho GTPases function as GDP-GTP regulated binary switches that are activated in response to extracellular stimuli. Activated Rho GTPases in turn associate with effectors that then stimulate cytoplasmic signaling networks [74]. Rho-specific guanine nucleotide exchange factors (RhoGEFs) promote GDP- GTP exchange and formation of active Rho-GTP [12], whereas Rho-specific GTPase activating proteins (RhoGAPs) accelerate hydrolysis of the bound GTP to stimulate formation of inactive Rho-GDP [75].

There are 83 human RhoGEFs, with Dbl family RhoGEFs comprising the largest group (69 members) [9, 12]. Dbl family RhoGEFs are characterized by a

pleckstrin homology (PH) regulatory domain. The DH domain may be highly or broadly specific for a subset of Rho GTPases. For example, Tiam1 is a specific activator of Rac, Asef is specific for Cdc42, whereas Vav is broadly active and can activate RhoA, Rac, Cdc42 and RhoG.

The significantly greater number of RhoGEFs relative to their GTPase substrates suggests highly redundant mechanisms for Rho GTPase activation. This apparent redundancy in activators is particularly striking for RhoA, where there are at least 25 Dbl RhoGEFs that can activate this Rho GTPase [12]. However, the multiple RhoGEFs for one Rho GTPase provides mechanisms where a specific Rho GTPase can be activated by divergent signals, leading to distinct spatio-temporal patterns of activation and signaling. This divergence in RhoGEF regulation and function is reflected by the otherwise highly divergent sequences that flank the DH-PH domains [74]. RhoGEFs are generally large proteins (>1000 amino acids), and often contain several domains involved in their localization, association with other proteins, regulation of GEF catalytic activity and Rho GTPase effector selectivity. For example, among all human RhoGEFs, only Ect2 contains tandem BRCT (BRCA1 C-terminal) domains that are normally found in proteins (e.g., BRCA1, TP53BP1) involved in DNA damage signaling, repair and coordination of cell cycle checkpoints [37, 76]. The BRCT domains may serve to regulate the RhoGEF catalytic activity and additionally can interact with phosphorylated proteins that dictate Ect2 subcellular localization [31, 77, 78].

Ect2 is also only one of two Dbl family RhoGEFs, aside from Net1 [79], that exhibits a nuclear localization in interphase cells [32]. Since Rho GTPases are membrane-associated and/or cytoplasmic, it has been proposed that this nuclear localization is an inactive repository for Ect2, since disruption of nuclear localization activates Ect2 transforming activity [36]. However, a recent study found that nuclear Ect2 maybe active [80], and thus, whether nuclear Ect2 has a function during interphase and distinct from cytokinesis remains unresolved. The best-known function of Ect2 involves its requirement for cytokinesis in vitro [65, 81]. In early mitosis (prometaphase) when the nuclear envelope is dissolved, Ect2 is released into the cytoplasm. As cells enter anaphase, Ect2 is recruited to the central spindle by the central spindlin protein complex comprised of Cyk4/MgcRacGAP and MKLP1/CHO1. The central spindle facilitates contractile ring formation, leading to cleavage furrow ingression and midbody formation. Ect2 is recruited to the midbody where Ect2 then activates RhoA to facilitate midbody abscission and completion of cytokinesis. Ectopic expression of a noncatalytic fragment of Ect2 that includes the BRCT domains caused inhibition of U-2 OS human osteosarcoma [32] or adenovirus-transformed HEK 293T human kidney neuronal-like [82] to complete cytokinesis, resulting in the accumulation of multinucleated cells. Similarly, microinjection of anti-Ect2 antibody into HeLa human cervical carcinoma cells also inhibited cytokinesis [32]. Subsequent studies using transient RNA interference suppression of Ect2 expression in HEK 293T [82] or HeLa [31, 83] cells also demonstrated a

requirement for Ect2 in cytokinesis in vitro. No assessment of mammalian Ect2 function in normal cells in vitro or in vivo has been described.

Although Ect2 was identified originally as an activated oncoprotein that was activated by truncation and loss of N-terminal sequences that include the BRCT domains and nuclear localization signals [19], no such truncated proteins have been detected in human cancers. Instead, recent studies have identified aberrant overexpression and mislocalization of full length Ect2 to the cytoplasm in glioblastoma and lung cancer tumor tissue and cell lines [38, 60, 62]. These studies used RNA interference to suppress Ect2 expression, which caused impaired lung tumor cell anchorage-independent growth and Matrigel invasion in vitro and reduced tumorigenic growth in vivo. Interestingly, in lung tumor cells, Ect2 suppression did not impair cytokinesis, indicating that Ect2 function in oncogenesis is distinct from that in cytokinesis [38]. Further support for this possibility was provided by the observation that constitutively activated Rac1 could rescue the loss of endogenous Ect2 and restore lung tumor cell growth [38]. This finding contrasts with previous studies that found that RhoA is the substrate critical for Ect2-dependent cytokinesis [65, 81].

Previous studies of mammalian Ect2 function have been done in established cell line studies in vitro. To evaluate the function of Ect2 in vivo, in normal cells and in the context of heterogeneous tissue, we generated both floxed (conditional) and Δfloxed (constitutive) knockout mice to assess the

consequences of Ect2 loss in development and normal cell proliferation. Constitutive loss of Ect2 causes embryonic lethality, with defective growth at the

late blastocyst stage. Utilizing mouse embryo fibroblasts derived from Ect2fl/fl conditional mice, we determined that loss of Ect2 completely impaired cell proliferation and migration in vitro, causing accumulation of cells in G2/M phase and formation of enlarged multi-nucleated cells. Surprisingly, expression of activated Rho GTPases failed to rescue the loss of Ect2 to restore cell proliferation or migration. Our observations provide further evidence for the highly unique function of this RhoGEF in normal cell physiology.

Methods and Materials

Vector construction: The targeting vector was based on a 5.3 kb genomic fragment from the ect2 gene encompassing exons 8 to 12 and surrounding sequences. This fragment, obtained from the C57Bl/6J RP23 BAC library, was modified by inserting the distal loxP site into intron 8 and the proximal loxP site including an FRT-flanked neomycin resistance gene into intron 7. A thymidine kinase (TK) cassette was inserted at the 3’ end of the genomic fragment.

Embryonic stem (ES) cell culture: The quality tested C57BL/6NTac ES cell line was grown on a mitotically inactivated feeder layer comprised of MEFs in Dulbecco’s modified eagle (DMEM) high glucose medium supplemented with 20% fetal bovine serum (FBS; PAN Biotech GmbH) and 1200 U/ml leukemia inhibitory factor (Millipore; ESG 1107). One x 107 ES cells and 30 µg of linearized DNA targeting vector were electroporated (Biorad Gene Pulser) at 240 V and 500

Resistant ES cell colonies with a distinct morphology were isolated on day eight after transfection and expanded in 96 well plates. Correctly recombined ES cell clones were identified by Southern blot analysis using external and internal probes and were frozen in liquid nitrogen.

Generation of mice: The animal study protocol was approved according to the German Animal Welfare Act (§ 8 (1) TierSchG) by the local authority. Mice were kept in the animal facility at TaconicArtemis GmbH in microisolator cages (Techniplast Sealsave). Feed and water were available ad libitum. Light cycles were on a 12:12 h light:dark cycle with the light phasing starting at 06:00 h. Temperature and relative humidity were maintained between 21 to 23°C and 45 to 65%, respectively. After administration of hormones, superovulated BALB/c females were mated with BALB/c males. Blastocysts were isolated from the uterus at dpc 3.5. For microinjection, blastocysts were placed in a drop of DMEM supplemented with 15% FBS under mineral oil. A flat tip, piezo actuated microinjection-pipette with an internal diameter of 12 - 15 µm was used to inject

10 to15 targeted C57BL/6NTac ES cells into each blastocyst. After recovery, eight injected blastocysts were transferred to each uterine horn of 2.5 days post coitum, pseudopregnant NMRI females. Chimerism was measured in chimeras (G0) by coat color contribution of ES cells to the BALB/c host (black/white). Highly chimeric mice were bred to i) C57BL/6-Tg(CAG-Flpe)2Arte females mutant for the presence of the Flp recombinase gene or to ii) C57BL/6- Gt(ROSA)26Sortm16(Cre)Arte females mutant for the presence of the Cre recombinase gene. This allowed detection of germline transmission by the

presence of black, strain C57BL/6, offspring (G1) and creation of i) selection marker deleted conditional (floxed) knockout mice or ii) constitutive knockout mice by Flp- or Cre-mediated removal, in one breeding step. Chimeras bred to iii) C57BL/6 wild-type mice resulted in germline offspring with selection marker in the Ect2 genomic locus described as targeted mice.

Genotyping of mice by PCR: Genomic DNA was extracted from 1 to 2 mm long tail tips using the NucleoSpin Tissue kit (Macherey-Nagel). Genomic DNA (2 µl) was analyzed by PCR protocol 1 in a final volume of 50 µl in the presence of 2.0 mM MgCl2, 200 µM dNTPs, 100 nM of each primer, and 2 U Taq DNA polymerase (Invitrogen) with the following primers: 1 = (5’-

GCACTCCAATTATGAAGCCAGAATGG-3’), 2 = (5’-

CAATATGTTGGGTAGAGAGATGGC-3’) and 3 = (5’-

TCCTCCGGGTGGACCAGAG-3’) detecting the presence of the wild-type allele (335 bp), targeted (413 bp), the conditional allele (413 bp) and the constitutive allele (498 bp). Following a denaturing step at 95°C for 5 min, 35 cycles of PCR

were performed, each consisting of a denaturing step at 95°C for 30 s, followed by an annealing phase at 60°C for 30 s and an elongation step at 72°C for 1 min.

PCR was finished by a 10 min extension step at 72°C. Amplified products were analyzed using 2% standard TAE agarose gels. A second PCR protocol for genotyping the Ectfl/fl mice used two primers: 3 = (5’- TCTGATCCACGTGATCATAG-3’), and 4 = (5’-CTGGCTT CATAATTGGAGTGC- 3’). A 420 bp wild-type fragment or a 520 bp mutant fragment was amplified with these two primers. For genotyping the blastocysts, a nested PCR protocol was

used. The outgrowths were scraped from the well with a micropipette tip and transferred to a PCR tube. The tubes were spun down and the supernatant was removed. The cells were resuspended in 5 µl of 400ng/µl Proteinase K/17 µM SDS and overlayed with mineral oil. The tubes were incubated for 1 h at 50°C, and then denatured at 99°C for 30 min. For the first PCR reaction, 45 µl of a PCR mix containing 25 pmol of external primers (5’-

CCCTCCAGGTTGAGAACTGCTACTAAG-3’ and 5’-

GCAGGCTGAGAGCAAGCCAGGAGA-3’). After amplification, 1 µl of this first PCR reaction was used for a second round of PCR reactions, which used PCR protocol 1 as described above.

Blastocyst outgrowth analyses: Timed breedings of ect2 +/- breeding pairs were set up and at E3.5, blastocysts were flushed from the uterine tract from the females. Blastocysts were placed in 20 µl of ES cell medium (DMEM, 15% FCS, 0.01% β-mercaptoethanol) in Nunc dish microwells (Nalgene,Palo Alto, CA) and grown under mineral oil in 5% CO2 incubator at 37°C. After seven days, the cultures were evaluated and scored for degree of outgrowth, photographed, and then genotyped by PCR.

Mouse embryonic fibroblasts cultures: Ect2fl/fl _{mice were crossed and}

at E15.5 days embryos were isolated from the female. Embryos were rinsed with PBS, and the head and dark red organs were removed from the culture. The tissue was minced and resuspended in a solution of PBS/trypsin/EDTA and incubated with gentle shaking at 37°C for 30 min. The cell/tissue solution was gently centrifuged and the supernatant was removed and the loose pellet was

resuspended in DMEM supplemented with 10% FCS. This solution was then filtered through a cell filter and then plated onto 150 mm tissue culture plates and grown in a 5% CO2 _{incubator at 37°C. After 18 h, the medium was changed to} fresh growth medium. MEFs were immortalized by trypsinizing the cells every three days at replating on a 100 mm tissue culture plate at a density for 1 X 103 cells/ dish. For knocking out Ect2 expression in the Ectfl/fl MEFs, cells were infected with recombinant adenovirus expressing Cre recombinase (Ad-Cre) with a green fluorescent protein expressing control adenovirus (Ad-GFP) used to assess Cre-specific activities (Gene Transfer Vector Core, University of Iowa).

Constructs: An expression vector for human Ect2 was generated using a human Ect2 cDNA sequence from an expression vector kindly provided by Dr. A. P. Fields (Mayo Clinic, Rochester, MN), which was subcloned into the FUGW lentiviral expression vector [84], a gift from Dr. Bryan Roth (University of North Carolina at Chapel Hill, Chapel Hill, NC). Mammalian expression vectors of wild- type human Rho GTPases [RhoA, Rac1 and Cdc42] were constructed by ligation of cDNA sequences into a pBabe-puro retrovirus expression vector [85]that introduces an N-terminal HA epitope tag. The constitutively active [RhoA(Q63L), Rac1(Q61L) and Cdc42(Q61L)] and fast-cycling Rho GTPases mutants [RhoA(F30L), Rac1(F28L) and Cdc42(F28L)] were created using QuikChange site-directed mutagenesis kit (Stratagene).

Western blot: To evaluate protein expression, four days after Cre infection, the MEFs were lysed, and analyzed by SDS-polyacrylamide gel electorphoresis and western blot analysis, with anti-Ect2 antibody (Santa Cruz

Biotechnology), anti-HA (Covance) and anti-β-actin (Sigma) as a loading control.

Additionally, we utilized a second Ect2 polyclonal antibody generated against KLH-conjugated linear peptide corresponding to C-terminal sequences in human Ect2 (Millipore).

Immunofluorescence: To visualize nuclei content, three days after Ad- Cre-infection the MEFs were plated onto coverslips. Twenty-four h after attachment, coverslips were rinsed with PBS, and fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min. The cells were stained for 5 min with 5 ng/ml 4',6-diamidino-2-phenylindole (DAPI) in PBS, washed 2-3 times with PBS and then mounted onto slides. The cells were visually analyzed for nuclei content on an Olympus BX61 upright fluorescence microscope. Images collected using Velocity (Perkin Elmer). To visualize polymerized F-actin, cells were stained with Alexa Fluor® 647 phalloidin (Invitrogen).

Flow cytometry: Cells were trypsinized, washed in PBS and then fixed in 70% ethanol and then stored at -20°C. The day prior to analysis the cells were washed in PBS and then stained with 0.5 ml of propidium iodine staining solution (1% Triton-X-100, 1 mg/ml PI, 100 mg/ml of RNAse A) overnight at 4°C. Data were collected on Beckman Coulter CyAn analyzer and ModFit (Becton Dickinson) was used for cell cycle analysis.

Results

Generation of Ect2 mutant mice. In order to evaluate the function of Ect2 in mammalian development we generated a conditional null mutation of the

mouse Ect2 locus by gene targeting. Our targeting strategy focused on exon 8, which encodes the N-terminal tandem BRCT domains, by flanking it with loxP sites (Figure 2-1A). Cre-mediated deletion resulted in a Δfloxed allele lacking

exon 8 that is a null mutation based on the complete loss of Ect2 protein expression as we describe below. Figure 2-1A shows a schematic diagram of the wild-type Ect2 allele, the targeting vector, the targeted allele after homologous recombination, and the resulting floxed (conditional) and Δfloxed (conditional null)

alleles.

Figure 2-1 Targeted disruption of the mouse Ect2 gene produces a null mutation

(A) Schematic diagram of the targeting vector, the wild-type allele, and the resulting Ect2 floxed allele (fl/fl) after FLP mediated deletion and the Ect2Δfloxed (null) allele after Cre mediated

Genotypic analysis of wild-type (+/+), heterozygous Δfloxed (+/-), mutant (null; mut) and floxed

(fl/fl) DNA by PCR. Primer pair 1 and 2 produced a 334 bp wild-type fragment. Primer pair 3 and 2 produced a 498 bp mutant fragment. Primer pair 3 and 4 produced a 535 bp fragment from the wild-type allele, and a 655 bp fragment from the floxed allele. Abbreviations used are: DNA isolated from heterozygous constitutive (Δfloxed) mice, +/- DNA; DNA isolated from wild-type

mice, +/+ DNA; DNA isolated from homozygous conditional (floxed) mice, fl/fl DNA; detection of the constitutive allele, Δfloxed; detection of the conditional allele,floxed); detection of the wild-type

allele, WT. (Data provided by Kauselmann, Schoor, Kuehn, Friedman)

Peri-implantation lethality of Ect2 mutant embryos. A ubiquitous Cre driver was used to generate constitutive null heterozygotes. Intercrosses of these Ect2+/- _{mice failed to produce any homozygous mutants at birth among 224}

offspring, suggesting that Ect2-/- is embryonic lethal (Table 2-1). Therefore, we performed Ect2+/- intercrosses as timed matings and genotyped embryos at progressively earlier stages of development. No Ect2-/- _{embryos were obtained at}

embryonic days (E) 15.5, 13.5, or 8.5 (Table 2-1). These results suggested that Ect2 is required for either pre-implantation or early post-implantation development.

Table 2-1. Genotypes of Weanlings and Embryos from Ect2 -/+ Intercrosses

(Data provided by Solski, Bultman, Cowley, and Van Dyke)

Genotype

Stage +/+ +/- -/-

At weaning 92 132 0

Embryos (13.5-15.5) 10 16 0

Embryos (8.5)a 5 5 0

a _{Examination of maternal uteri exhibited empty deciduas, suggesting peri-implantation}

lethality.

To investigate the peri-implantation stage in more detail, we performed additional Ect2+/- intercrosses, isolated blastocysts at E3.5, and analyzed their outgrowth ex vivo [86]. Homozygous Ect2-/- blastocysts had a morphologically

normal trophectoderm (TE) and inner cell mass (ICM) (Figure 2-2). We cultured 125 blastocysts for five days and then scored the resulting outgrowths and determined their genotypes. After this culture period, 84% of wild-type outgrowths and 92% of Ect2+/- outgrowths were scored as normal based on the presence of both TE and extensive ICMs (Figure 2-2, Table 2-2). In contrast, no Ect2-/- outgrowths were scored as normal. Instead, 14% of homozygous blastocysts failed to hatch from their zona pellucida and the remaining 86% were scored as abnormal based on the ICM not being viable. These results indicate Ect2 is required for peri-implantation development.

Figure 2-2 Ect2-/- blastocysts display abnormal growth in vitro